Prostate-specific membrane antigen (PSMA) as a potential target for molecular imaging and treatment in bone and soft tissue sarcomas

Bone and soft tissue sarcomas are a group of rare malignant tumours with major histological and anatomical varieties. In a metastatic setting, sarcomas have a poor prognosis due to limited response rates to chemotherapy. Radioligand therapy targeting prostate-specific membrane antigen (PSMA) may offer a new perspective. PSMA is a type II transmembrane glycoprotein which is present in all prostatic tissue and overexpressed in prostate cancer. Despite the name, PSMA is not prostate-specific. PSMA expression is also found in a multitude of non-prostatic diseases including a subgroup of sarcomas, mostly in its neovascular endothelial cells. On PET/CT imaging, multiple sarcomas have also shown intense PSMA-tracer accumulation. PSMA expression and PSMA-tracer uptake seem to be highest in patients with aggressive and advanced sarcomas, who are also in highest need of new therapeutic options. Although these results provide a good rationale for the future use of PSMA-targeted radioligand therapy in a selection of sarcoma patients, more research is needed to gain insight into optimal patient selection methods, PSMA-targeting antibodies and tracers, administered doses of radioligand therapy, and their efficacy and tolerability. In this review, mRNA expression of the FOLH1 gene which encodes PSMA, PSMA immunohistochemistry, PSMA-targeted imaging and PSMA-targeted therapy in sarcomas will be discussed.


INTRODUCTION
Sarcomas are a diverse group of malignant tumours that arise from connective tissues, such as bone, cartilage, fat, muscle and blood vessels. These tumours can occur in many different anatomic locations and currently, over 70 histological subtypes are defined. 1 Sarcomas can be divided into two broad categories: soft tissue sarcomas and bone sarcomas, with an estimated incidence of 4.7 per 100,000 /year and 0.8 per 100,000 /year, respectively. 2 Together they represent less than 1% of all new cancer cases. 3,4 Due to its rarity and its histological and anatomical heterogeneity, diagnosis and optimal tumour management are challenging. Individual treatment plans of sarcoma patients should therefore always be made in multidisciplinary teams of sarcoma reference centres. 5,6 The golden standard for localized intermediate and high-grade sarcomas is complete surgical resection with wide and microscopically tumour-free (R0) margins. Selected patients, e.g. in case of high-risk lesions, can be treated additionally with (neo)adjuvant radiotherapy and/ or chemotherapy to improve clinical outcomes. Although complete removal of the tumour is pursued, unfortunately around 30-50% of patients, also depending on tumour characteristics, still develop local recurrences and/or metastases after primary treatment. [7][8][9][10] In addition, at least 14-17% of sarcoma patients have distant metastases at presentation. 11 Standard of care in patients with metastatic sarcomas consists of cytotoxic chemotherapy, with doxorubicin being the first choice in most sarcoma types. 5,6 With the administration of doxorubicin, in combination with or without ifosfamide chemotherapy, treatment response rates of approximately 25% are reached in advanced soft tissue sarcoma 12,13 and even lower in bone sarcomas. Due to these low response rates to chemotherapy, the five-year survival rates in metastatic soft tissue sarcoma and bone sarcoma are 17 and 31%, respectively. 14 This shows that there is a high need for new effective treatment options that can decrease burden of disease and increase survival, especially in sarcoma patients with advanced disease.
Prostate-specific membrane antigen (PSMA) may offer a new perspective for sarcoma patients. Despite the name, PSMA is not prostate cancer-specific. PSMA, also known as glutamate carboxypeptidase II, is a type II transmembrane glycoprotein which consists of 750 amino acids and is encoded by the FOLH1 (folate hydrolase 1) gene. 15 In prostatic tissue, PSMA is expressed in the secretory epithelial cells, and PSMA is overexpressed in prostate cancer. Further upregulation of PSMA expression is seen in more advanced prostate cancers and PSMA was shown to be an independent predictor of poor prognosis. 16 Although the exact mechanisms are unknown, PSMA is associated with the activation of PI3K/AKT and cAMP/PKA pathways, which are involved in cell proliferation. 17,18 The expression pattern of PSMA has made it a well-established target for molecular imaging in prostate cancer. In the last decade, PSMA-targeting PET/CT scans have found their way into clinical practice for primary staging of high-risk prostate cancer patients and in case of biochemical recurrence after primary treatment. 19 Furthermore, PSMA targeting ligands have been labelled with therapeutic nuclides such as lutetium-177 or actinium-225, and these radioligand therapies have achieved beneficial effects in advanced prostate cancer patients with acceptable toxicity. 20,21 Interestingly, PSMA has been found in the tumour-associated neovascular endothelial cells of a wide variety of other tumours besides prostate cancer, such as renal cell carcinoma, glioblastoma, hepatocellular carcinoma, thyroid cancer, lung cancer, breast cancer, and sarcoma. 22 Since PSMA-targeted radioligand therapy (PSMA-RLT) has demonstrated promising results in patients with prostate cancer, the question arises whether this therapy could also have a beneficial effect in other PSMA-positive tumours, such as sarcomas.
This review aims to give an overview of the available literature about the possible role of PSMA in sarcomas. mRNA expression of the FOLH1 gene, immunohistochemical PSMA expression, PSMA-targeted imaging, PSMA-targeted therapy and the possible future perspectives for sarcomas will be discussed. This resulted in 28 articles, of which 15 were relevant for the topic of this review. The excluded articles were either not about PSMA in sarcomas (n = 11), or were reviews that cited already included articles (n = 2). The same search strategy was used in Scopus and Web of Science, which did not add other relevant articles. Also, no extra articles were found while reading the included articles and their references. PubMed, Scopus and Web of Science were last checked for new articles on 05-12-2022. mRNA expression of the FOLH1 gene in sarcomas PSMA is encoded by the FOLH1 gene, which is localized on chromosome 11p11-p12 and contains 19 exons. 23 As part of the large-scale Pan-Cancer analysis project from The Cancer Genome Atlas Research Network, the FOLH1 mRNA expression levels were determined in over 10.000 tumours from 32 different tumour types, together with a wide variety of other genetic and clinical data. Figure 1 shows the results from this analysis, obtained from cBioPortal. 24,25 The FOLH1 mRNA expression levels were calculated by the logarithmic transformation of FOLH1 abundance estimates using RSEM. 26 After sortation of FOLH1 mRNA expression levels by median, sarcomas were 12 th of the 33 tumour types. There still was a notable difference between the FOLH1 mRNA expression levels in prostate carcinomas and all other carcinomas, which can be seen in Figure 1.
The Pan-Cancer Atlas also contained overall survival and progression-free survival data. Within the leiomyosarcoma group, higher FOLH1 mRNA expression levels correlated significantly with shorter progression-free survival (Spearman correlation = −0.25, p = 0.015, Figure 3). However, R 2 was 0.06, so only a small percentage of the variation could be attributed to the FOLH1 mRNA expression levels. No such correlation was seen with overall survival. In the other sarcoma subtypes, no correlation was found between FOLH1 mRNA expression levels and progression-free survival or overall survival.

PSMA immunohistochemistry in sarcomas
For determining PSMA expression in formalin-fixed paraffinembedded (FFPE) tissue samples, immunohistochemistry is the designated technique. Up until now, four articles have investigated immunohistochemical PSMA expression in sarcomas.  27 of which 599 were sarcomas. PSMA expression was found to be positive in 151 of 779 soft tissue and bone tumours (19.4%). Similar to other non-prostate tumours, this PSMA expression was found in the tumourassociated neovasculature and was more frequent in sarcomas compared to soft tissue and bone tumours with benign or intermediate biological potential. Strong PSMA expression, defined as moderate staining (readily apparent at 40x magnification) in >5% of the neovasculature or any strong staining of the neovasculature, was found in 43 of 779 soft tissue and bone tumours (5.5%). Table 1 lists the sarcoma subtypes of which over 20% of analysed tumour samples showed any PSMA expression. The sarcoma subtypes with the highest frequency of PSMA-positivity were pleomorphic rhabdomyosarcoma (60%), synovial sarcoma (56%) and pleomorphic liposarcoma (50%). These were also the sarcoma types with the highest number of tumours with strong PSMA expression (40%, 38 and 20%, respectively). As for bone sarcomas, 106 Ewing sarcomas were investigated, of which six were PSMA-positive and none showed strong PSMA expression. No other bone sarcomas were included.
Zeng et al. investigated PSMA expression in osteosarcomas. 28 After immunohistochemical analysis, 21 of 45 osteosarcomas (47%) demonstrated PSMA reactivity in the tumour-associated neovasculature, not in the tumour cells. Interestingly, PSMA expression was significantly associated with tumour size (p = 0.042), the presence of pulmonary metastasis (p < 0.001) and a worse 5 year survival rate (36.6% vs 63.2%, p < 0.05). These findings support the hypothesis that PSMA expression is associated with worse clinical outcome. PSMA expression was not associated with age, gender and location. In two other studies, sarcomas were studied as part of a larger tissue sample cohort. Chang et al. studied 20 benign and 12 malignant tissue types, including seven soft tissue sarcomas. 29 Of these, six turned out PSMA-positive. No further histological information on these soft tissue sarcomas is described. Coskun et al. investigated PSMA expression, along with STAT3 and VEGF expression, in 25 malignant peripheral nerve sheath tumours (MPNST). 30 None of the 25 MPNSTs were PSMA-positive.
It is important to take into account that up until now, a wide variety of monoclonal antibodies (mAbs) have been developed to use for PSMA immunohistochemistry and that in the previously described articles, several different mAbs are used ( Table 1) . The 7E11 mAb was the firstly used anti-PSMA antibody, which binds to the intracellular epitope of PSMA. More recently developed mAbs, such as mAb 3E6, used by Heitkötter et al., 27 often bind to the extracellular epitope of PSMA. Chang et al. 29 compared two mAbs that bind to the intracellular PSMA domain (7E11 and PM2J004.5) to three other mAbs that bind to the extracellular PSMA domain (J591, J415 and PEQ226.5). All five anti-PSMA  Table 2 describes each of these case reports.
First of all, three liposarcomas have shown PSMA uptake; a well-differentiated, dedifferentiated and pleomorphic liposarcoma. [31][32][33] It is remarkable that in the dedifferentiated liposarcoma, PSMA seemed to differentiate between the lipomatous and non-lipomatous regions, where the latter was PSMApositive. The highest tumour-to-background ratio was seen on the PSMA PET/CT scan of pleomorphic liposarcoma (SUV max = 13). Plouznikoff et al. and Mathew et al. reported two patients with PSMA-positive undifferentiated pleomorphic sarcoma in the left obturator muscle and the right posterior chest wall, respectively, one of which was likely radiation-induced. 34,35 As for bone sarcomas, one Ewing sarcoma and two osteosarcomas with PSMA uptake have been described. [36][37][38] Two of them were females, for which the PSMA PET/CT scans were specifically made to assess the amount of PSMA-tracer binding in the tumour, while in all other case reports the PSMA-avid sarcomas were incidental findings in patients with prostate cancer. In one patient with osteosarcoma, PSMA could differentiate between areas of fibrous dysplasia and areas of malignant transformation to osteosarcoma in the skull bones. When looking at sarcomas in a metastatic setting, one of the osteosarcoma patients and additionally one patient with angiosarcoma and two patients with leiomyosarcoma had multiple PSMA-avid metastases. [39][40][41] Interestingly, especially in these patient cases high PSMA uptake with high SUV max values were reported. The highest SUV max values were described in high-grade osteosarcoma, localized in the sternum and multiple lung, bone and liver metastases (SUV max values of 10.6-35.1). An example of one of our own patients with PSMA-avid metastatic sarcoma is shown in Figure 4.
The potential pitfall of PSMA-targeted PET/CT imaging is that not all PSMA-tracer uptake besides physiological uptake can be attributed to malignant lesions. Occasionally, PSMA expression and PSMA-tracer uptake are seen in benign neoplasms, such as haemangiomas and schwannomas. 22,27 Without careful interpretation, such lesions might be easily mistaken for metastases. [42][43][44][45][46] It is important to take this possibility into account while interpreting PSMA PET/CT scans of sarcoma patients, as this would otherwise lead to false-positive findings.

PSMA-targeted therapy in sarcomas
While for prostate cancer PSMA-RLT is still often given in a research setting, already two case reports have been published describing the administration of 177 Lu-PSMA-RLT to a patient with metastasized leiomyosarcoma, both also described in Table 2. 40,41 The first patient was a 50-year-old female who was diagnosed with a leiomyosarcoma of the vena cava inferior in 2015 (TNM pT2pN0(0/9)pR0 FNCLCC Grade 1). 40 In 2016, the patient underwent selective internal radiotherapy for several liver metastases. In 2017, routine diagnostics revealed new metastatic lesions in bone, lung and muscle. As routine systemic therapies were denied by the patient, a diagnostic PSMA PET/CT scan was performed to assess the feasibility of PSMA-RLT. Because of low SUV max values, re-evaluation with PSMA PET/CT was done one year later, which showed clear progression of disease with multiple new lesions and increased SUV max values. The lesions in both lungs, liver, left gluteus maximus muscle, right vastus lateralis muscle and multiple bone lesions were treated with one application of 6.0 GBq [ 177 Lu]Lu-PSMA-617. The radionuclide therapy was well tolerated by the patient, but intratherapeutic whole body scans revealed moderate PSMA uptake in the left gluteus maximus muscle and left os ilium and weak uptake in all other lesions. Therefore, no other [ 177 Lu]Lu-PSMA-617 cycles were given. Three months later, further progression of the disease was seen. The images of this patient are shown in Figure 5.
Another female patient was diagnosed with uterine leiomyosarcoma with peritoneal metastases in 2018, in her mid-50s. 41 After several palliative chemotherapy treatments, tumour progression was observed. Due to the presence of tumour-infiltrating lymphocytes and a lack of standard treatment options, off-label nivolumab treatment was initiated in February 2021. Four months later, the tumour growth rate was +36.5% per month (compared to +23.8% per month before nivolumab was started).
To test the feasibility of PSMA-RLT, a PSMA PET/CT scan was performed, which showed PSMA uptake in lung, adrenal and peritoneal lesions. Two cycles of [ 177 Lu]Lu-PSMA-I&T with a two-month interval were administered in combination with  nivolumab. Four months after the last dose, the tumour growth rate reduced to +11.3% per month. The lung lesion with the highest PSMA uptake (SUV max = 8.9) showed a considerable size reduction. At this time, nivolumab treatment was stopped. The administered dose of [ 177 Lu]Lu-PSMA-I&T and its tumour retention time, however, were not described in this case report.

Future perspectives
To assess the feasibility of PSMA-targeted imaging and therapy in patients with sarcomas, it is important to know whether or not PSMA expression is seen in the tumour, and which factors determine effective PSMA-RLT. Previously published literature reveals that not all sarcomas, but definitely a subgroup of sarcomas show (strong) PSMA expression in their tumour-associated neovasculature, and (high) PSMA-tracer uptake on PET/CT imaging. These results provide a rationale that PSMA-RLT can be successful in a selection of sarcoma patients.
In prostatic tissue, PSMA expression is upregulated in case of malignant transformation and is further upregulated in more advanced stages of disease. Several studies show that PSMA expression is an independent prognostic factor in prostate cancer patients, associated with worse survival. 16 Similar patterns can be observed in sarcomas. PSMA immunohistochemistry studies revealed that sarcoma types had significantly higher PSMA expression compared to tumour types with benign or intermediate biological potential. 27 Furthermore, in osteosarcomas, PSMA expression was significantly associated with tumour size, the presence of pulmonary metastasis and a worse 5 year survival rate. 28 In previously published case reports of PSMA-tracer uptake in sarcomas on PET/CT imaging, PSMA was able to differentiate between dedifferentiated liposarcoma and lipomatous regions 32 and between osteosarcoma and fibrous dysplasia, 37 showing visibly higher PSMA-tracer uptake in the malignant lesions. Although not every case report has described SUV max values, metastasized sarcomas seem to have more intense PSMA-tracer uptake compared to non-metastasized sarcomas. 38,40 Lastly, one patient with metastasized leiomyosarcoma who received multiple PSMA PET/CT scans showed a noticeable increase in SUV max values after one year of disease progression. 40 These observations suggest that if present, PSMA expression seems to be highest in patients with aggressive and/ or advanced sarcomas, similar to prostate cancer. These are also the patient groups that are in highest need of new therapeutic options and could benefit the most from PSMA-RLT.
Currently, there is no evidence for an optimal selection method yet to determine whether a sarcoma patient is likely to respond to PSMA-RLT. The most evident option seems to select eligible patients by using PSMA immunohistochemistry on biopsy or resection material to identify tumours with high PSMA expression. In prostate cancer, the percentage of PSMA-expressing tumour cells correlates significantly with SUV max on PSMA PET/CT imaging. 47 However, no studies have investigated the correlation between PSMA immunohistochemistry and SUV max on PSMA PET/CT imaging in sarcomas yet. Therefore, no definition of 'high PSMA expression' , enough for significant PSMA-tracer uptake in sarcomas on PSMA PET/CT imaging, is available. As sarcomas have mainly shown PSMA expression on the neovascular endothelial cells instead of on the tumour cells itself (with some rare exceptions), the radiotracer uptake is expected to be less than in prostate carcinoma. However, the case reports of e.g. Militano et al., Can et al. and Jüptner et al. show that high SUV max values over 12 can be reached in sarcomas, which generally is considered enough to explore the possibility of PSMA-RLT. 48,49 Additionally, several case reports that describe 177 Lu-PSMA-RLT treatment in other non-prostatic malignancies with neovascular PSMA expression, such as glioblastoma multiforma, show that good tumour retention (in a lesion with a SUV max of 10.3) and a significant reduction in tumour size and symptoms can be achieved, with acceptable toxicity. 50,51 It is also important to consider that a direct correlation between PSMA-tracer uptake on PSMA PET/CT imaging and the intratumoural dose after PSMA-RLT is not self-evident, as the 6.0 GBq of [ 177 Lu]Lu-PSMA-617 that was administered to a patient with metastasized leiomyosarcoma, showed limited retention in the tumour 24 h after injection compared to the diagnostic 68 Ga-PSMA PET/CT scan that was obtained beforehand.
Although the currently available literature suggests that there might be a selection of sarcoma patients that could benefit from PSMA-RLT, it should be realized that this suggestion is based on a limited amount of patient studies and a few number of clinical cases. For a successful treatment with PSMA-RLT, two factors are of great importance: 1) the achieved intratumoural radioactive dose, which is influenced by multiple factors such as the total administered radiation dose, the tumoural reachability, the radiotracer's affinity towards PSMA, its retention time and BJR PSMA as a target for molecular imaging and treatment in sarcomas the level of PSMA expression, and 2) the radiosensitivity of the tumour. Further investigation is necessary to gain knowledge about each of these aspects regarding sarcoma patients, e.g. by comparing different PSMA-targeting antibodies and tracers, evaluating their tumour dosimetry and assessing PSMA-RLT efficacy and toxicity profiles with different doses. However, first of all, more insight is needed into what tumour characteristics predict significant intratumoural binding of PSMA-ligands. For this, currently, one trial is recruiting patients to compare PSMA immunohistochemistry in biopsy material with, in case of high PSMA expression, PSMA-tracer accumulation on PET/ CT imaging ( clinicaltrials. gov, NCT05522257). Furthermore, in end-stage metastatic prostate cancer, 177 Lu-and 225 Ac-labelled PSMA have proven to prolong overall and progressionfree survival with acceptable toxicity. 20,21 Recently, the first results on 89 Zr-labelled PSMA ligands in prostate cancer have been published, having the advantage of a longer half-life and allowing acquisition at later time points after injection. 52,53 This might result in higher tumour-to-background ratios, better assessment of tumour retention and improved patient selection for PSMA-RLT compared to 18 F or 68 Ga. Developments in this field and the increasing knowledge about the role of PSMA in different neoplasms should be closely monitored and used for the rationale regarding PSMA-RLT in sarcoma patients as well.

CONCLUSION
Strong PSMA expression and good PSMA-tracer accumulation are observed in a selection of sarcoma patients, which seems to be more prevalent in aggressive and advanced sarcomas. These patient groups are also in highest need of new therapeutic options, as their 5-year survival rates are low. The results from previous literature have laid the foundation for further research that is needed to investigate the possible efficacy of PSMA-RLT in sarcomas and how eligible sarcoma patients could be selected in a reliable way. Hopefully, additional insights will be given by the currently recruiting prospective study ( clinicaltrials. gov, NCT05522257).